Guidance

Annexe 2b. Screening criteria for in vivo papers

Published 11 October 2024

Tier 1. Nanomaterial and generic study design

When assessing papers based on nanomaterial characteristics and generic study design, all papers were scored against the criteria outlined in Table 1. If sufficient data were available in the paper, a score of 1 was given. For example, a score of 1 was awarded if sufficient data were presented on nanoparticle (NP) size and shape or exposure duration and observation time points. More weight was placed on some criteria, such as availability of data on positive controls and the number of animals per group, and hence these were given a score of 2.

Papers with a total score of 7 out of 10 and above proceeded to tier 2 of screening and were further evaluated by assessing the basic genotoxicity study design (see below).

Table 1. Tier 1 assessment criteria for nanomaterial characteristics and generic study design of in vivo genotoxicity studies on TiO₂

NM characteristics	test material and crystalline form (score = 2) particle size (score = 1) particle shape (score = 1) size at the start or at the end of the exposure period (score = 1) duration of exposure as well as time-points of observations (score = 1)
Study design	use positive controls (where appropriate) (score = 2) number of animals per group (score = 2)

Tier 2. Generic genotoxicity study design

When assessing papers based on generic genotoxicity study design, papers were scored against the criteria outlined in Table 2. As with the assessment of nanomaterial characteristics, some characteristics of the genotoxicity study design, including use of positive and negative controls and number of animals, were given a higher weighting. Papers with a total score of 12 out of 16 and above went to tier 3 and were further evaluated by expert review by Committee on Mutagenicity (COM) members, in which the detailed genotoxicity study design was assessed.

Table 2. Tier 2 assessment criteria for genotoxicity study design of in vivo genotoxicity studies on TiO₂

NM characteristics	identification of test material (score = 1) source or origin of test material (score = 1) purity (concentration) of test material (score = 1) doses administered or concentration in exposure media (score = 1) test medium or vehicle (score = 1)
Study design	species (score = 1) route of administration (score = 1) frequency and duration of exposure, and timepoints of observations (score = 1) use of negative controls (score = 2) use of positive controls (score = 2) numbers of animals (score = 2) study methods described (score = 1) statistical analysis (score = 1)

Tier 3. Detailed genotoxicity study design

When assessing papers based on the detailed genotoxicity study design, data on the criteria outlined in Table 3 was collated. The data was assessed using the assessment criteria listed below, using expert judgement.

Table 3. Tier 3 assessment criteria for genotoxicity study design of in vivo genotoxicity studies on TiO₂

Nanomaterial characteristics	primary and secondary size
Nanomaterial dispersion	method and surfactant
Test system	animal model, species, strain and sex number of animals or group exposure route dosing regime sampling time dose range number of cells scored tissue standard test system
Cytotoxicity assessment	cytotoxicity test used extent of cytotoxicity at genotoxic dose
Controls	negative control (background level) positive control level of increase over background
Cellular or target tissue uptake
Mechanism of action data
Results
Opinion on study quality and validity of approach

Exclusion criteria

Expert judgement was used to assess the quality and interpretation of the genotoxicity studies by noting a number of assessment criteria.

Only assays with Organisation for Economic Co-operation and Development (OECD) guidelines were included in the assessment, including the micronucleus (MN) assay (OECD TG474), chromosomal aberration (CA) assay (OECD TG475), Comet assay (OECD TG489), transgenic rodent somatic and germ cell gene mutation assays (OECD TG 488) and the phosphatidylinositol glycan class A gene (Pig-a) mutation assay (OECD TG470). The γ-H2AX assay and oxidative damage markers were also included for mechanistic information. Other assays were excluded from further evaluation.

Other exclusion criteria included the lack of positive controls, inadequate numbers of animals used, inappropriate sampling times, inadequate number of cells scored and high spontaneous levels of damage.

The studies were assessed according to the exclusion criteria and, based on the results, were classified as red, amber or green (RAG rating). Green indicates good robust studies without major deficiencies identified; Amber indicates studies considered sufficient for assessment, but with noted deficiencies; and Red indicates studies with significant deficiencies in procedural descriptions or protocols meaning that they were not of sufficient quality for use in the assessment of genotoxicity of TiO₂.

Application of the exclusion criteria listed above automatically led to some studies being graded as red (RAG rating) and these were not further assessed.

Overall, from a total of 53 papers that were initially assessed, six papers were categorised as green or amber and were considered to be relevant and of sufficient quality for use in the in vivo genotoxicity assessment of TiO₂ (Figure 1).

Figure 1. Summary of number of papers assessed in tier 1, 2 and 3

The 6 papers (reporting 12 assays) are summarised below together with a brief summary of the COM opinion for each paper. An overall summary draws a conclusion on the potential in vivo genotoxicity of TiO₂.

Two of the 12 assays were considered the most robust and were categorised as green, and 10 assays were categorised as amber. The number and type of assay in each category is shown in Table 4.

Table 4. Number and type of genotoxicity study classified as green, amber or red

Test	Green category	Amber category	Red category
MN	2	3	9
CA		1	1
Comet		1	15
hprt			1
Pig-a mutation		3
gpt mutation		1
γ-H2AX		1	2

Note: some papers assessed several endpoints.